Loop diuretics inhibit detubulation and vacuolation in amphibian muscle fibres exposed to osmotic shock

The effect of loop diuretics at concentrations known to influence cellular water entry coupled to Na-K-Cl co-transport, upon the vacuolation and detub ulation following osmotic shock, was investigated in amphibian skeletal mus cles. These were exposed to a glycerol-Ringer solution (18 min), an isotoni c Ca2+/Mg2+ Ringer solution and cooling. Adding bumetanide (1.0 and 2.0 mu M) to these solutions sharply reduced the incidence of detubulation, assess ed by abolition or otherwise of action potential after-depolarisations, fro m 93.9 +/- 4.7% (n = 6) to 5.0 +/- 1.1% (n = 4: mean +/- SEM: 2.0 mu M bume tanide). It dramatically reduced the number and fraction of muscle volume o ccupied by tubular vacuoles, measured using confocal microscopy, from 60.3 +/- 4.3% (n = 10) to 9.0 +/- 1.1% (n = 35). The incidence of large horserad ish peroxidase-lined tubular vacuoles, viewed using electronmicroscopy, sim ilarly was reduced with 2 mu M bumetanide in the glycerol-Ringer solution. Bumetanide acted through cellular volume adjustments early in the detubulat ion protocol. Thus, it exerted its maximum effect when added to the glycero l-Ringer, rather than the Ca2+/Mg2+ Ringer solution. Furthermore, whereas f ibre diameters measured using scanning electron microscopy returned to norm al during glycerol treatment relative to those of control fibres left in is otonic Ringer, addition of 2.0 mu M bumetanide in the glycerol Ringer left markedly smaller fibre diameters. Finally equipotent concentrations of the chemically distinct loop diuretics, furosemide and ethacrynic acid similarl y influenced detubulation. These findings implicate Na-K-Cl co-transport in the water entry into muscle fibres that would be expected following introd uction of extracellular glycerol. This might then enable the subsequent Na- K-ATPase dependent water extrusion that produces the tubular distension (va cuolation) and detachment (detubulation) following glycerol withdrawal, phe nomena also observed in muscular dystrophy.